3-HP (3-hydroxypropionic acid, C3) is an isomer of lactic acid (2-hydroxypropionic acid) and it has a carboxylic acid group and a hydroxyl group at both ends thereof, and thereby it is a useful material capable of being converted into various chemicals such as 1,3-propanediol, acrylic acid, acrylamide, a polymer, and the like. Actually, due to the above mentioned reason, 3-HP was selected as one of the promising chemicals that can be produced from biomass by the U.S. Department of Energy in 2004. Particularly, acrylic acid could be a major applied form of 3-HP, as highly marketable material used in a coating material, an adhesive, an additive, a diaper, or the like. 3-HP may be theoretically produced from various biomass such as glucose through fermentation at a yield of 100%, and a fermentation process using microorganisms is suitable for satisfying the demand for an eco-friendly and renewable material.
There is yet no case of commercial production of 3-HP using biomass, but research into various methods has been conducted, and the securing of an economical 3-HP producing strain is emerging as a major obstacle. Bacteria are known as representative microorganisms producing organic acids and widely used in industry such as a food industry, or the like. However, there are disadvantages for applying production of organic acid using industrial bacteria such as E. coli to large-scale chemical industry such as 3-HP production. As the production amount of organic acid is increased, a hydrogenated form of acid is increased and acidity is increased (pH is decreased), and thereby activity of most of the E. coli is decreased. In the case of producing an organic acid at a high concentration, bacteria require a base such as sodium hydroxide (NaOH) and ammonium hydroxide (NH4OH) for maintaining a neutral pH. This causes an increase in the cost of the fermentation process depending on an injected base, makes an extraction and separation process difficult, or significantly increases the cost.
In a recent case of application of producing organic acid to chemical industry, an organic acid is produced from glucose using yeast. Yeast has high resistance against an organic acid as compared to bacteria, thus the activity of the yeast is not significantly inhibited even at a high acidity (low pH) making yeast as more suitable host for producing the organic acid. Particularly, yeast has been conventionally widely used as an industrial biocatalyst for producing spirits, industrial ethanol, or the like, and may be mass-cultured and may not be contaminated with bacteriophages, such that applicability of the yeast in the chemical industry is more excellent as compared to bacteria. Yeast has advantages for producing an organic acid, but there are several disadvantages for using the yeast to produce an organic acid such as 3-HP. First, it is more difficult to genetically modify yeast as compared to bacteria, and in order to express a specific metabolic enzyme, the sub-cellular location for expression of the metabolic pathway along with the specific metabolic enzyme should be identified due to the shape of the cells divided into intracellular structural bodies such as mitochondria, peroxisomes, or the like, unlike bacteria. For example, a representative metabolic intermediate such as acetyl-CoA is mainly produced in the mitochondria in yeasts. However, if a target product is produced in the cytosol, a method for producing acetyl-CoA in the cytosol is also required. In addition, since there is large number of different yeast in the fungal kingdom and all of the yeast do not satisfy requirements for high productivity, resistance against an organic acid and massive cultivation, a host suitable for producing the organic acid should be effectively selected.
It is known that some yeast effectively produce ethanol from glucose, which is a hexose, and some yeast species also produce an organic acid. At the time of modification for producing a target product using microorganisms, it is important to maintain an entire balance of oxidation and reduction for a metabolic reaction, and also, a metabolic reaction of fermenting ethanol from glucose is a suitably maintained reaction. Even in the case of genetically modifying yeast to produce an organic acid, the balance as described above should be appropriately maintained, and in a case of producing lactic acid through modification of yeast, balanced introduction of lactic acid dehydrogenase (LDH) of another reduction reaction for complementing a reduction reaction of producing ethanol from acetaldehyde is important.
It is known that a small amount of 3-HP is produced in a small number of microorganisms such as Chloroflexus aurantiacus, and 3-HP is partially formed from a decomposition process of dimethylsulfoniopropionate in microorganisms such as Alcaligenes faecalis or a decomposition process of uracil in yeast. Research for 3-HP metabolic pathways and the corresponding enzymes have been conducted through discovery of 3-HP present in nature as described above, and based on the research, recently, research for a technology of producing 3-HP or a PHA, which is a polymer form of 3-HP, by introducing a gene required for 3-HP biosynthesis in E. coli has been conducted. In addition, in order to maximize productivity and production yield of 3-HP which is only present as a metabolic intermediate or produced only at a small amount in nature, technologies such as a metabolic engineering technology, a systems biology technology, a synthetic biology technology, or the like, have to be utilized.
According to the development of the metabolic engineering technology, it becomes possible to predict production pathways of various materials using microorganisms, and a pathway for producing 3-HP from glucose may be roughly divided into an acryloyl-CoA pathway, a β-alanine pathway, a malonyl-CoA pathway, and a glycerol pathway depending on metabolic intermediates.
The acryloyl-CoA pathway means a metabolic pathway of converting pyruvate or phosphoenol pyruvate (PEP) obtained from the glycolysis of glucose into acryloyl-CoA via lactate or β-alanine and then converting the acryloyl-CoA into 3-HP through a hydration reaction and a reduction reaction (pyruvate or PEP→lactate or β-alanine→acryloyl-CoA→3-HP). Acryloyl-CoA is a metabolite observed during the decomposition process of propionic acid, and since the Gibb's free energy value for formation of the metabolite is positive, a forward reaction is an unfavourable reaction. In addition, substrate specificity of acryloyl-CoA thioesterases is low, such that the acryloyl-CoA pathway is not suitable as the metabolic pathway for mass-producing 3-HP.
The β-alanine pathway means a metabolic pathway of converting pyruvate or oxaloacetate into amino acid by a transamination reaction and finally conversion into 3-HP via β-alanine by a transamination reaction (pyruvate or oxaloacetate→amino acid→β-alanine→3-HP; US 2012/0135481A1). Since the transamination reaction of β-alanine to 3-HP proceeds via malonate semialdehyde which is highly toxic to microorganisms, a 3-HP dehydrogenase having a high activity is required. In addition, generally, since the transamination reaction forms a radical form of an amino acid molecular structure in a steady-state, an enzyme of this reaction has a structure for alleviating reactivity of the radical. Since this radical has strong reactivity with oxygen, for a smooth transamination reaction, anaerobic conditions or a coenzyme for stabilizing radical molecules are essentially required.
The malonyl-CoA pathway is a metabolic pathway of converting acetyl-CoA into malonyl-CoA by carboxylation and then converting malonyl-CoA into 3-HP by a reduction reaction (acetyl-CoA→malonyl-CoA→3-HP), and the glycerol pathway is a metabolic pathway of converting glucose into glycerol, converting glycerol into 3-hydroxypropionaldehyde by a dehydration reaction, and then converting 3-hydroxypropionaldehyde into 3-HP (glucose→glycerol→3-hydroxypropionaldehyde→3-HP). Since the malonyl-CoA pathway and the glycerol pathway proceed through an intermediate generally produced by microorganisms such as E. coli, or the like, these pathways have been mainly studied as the 3-HP production pathway (US 2013/0071893 A1). Since malonyl-CoA may be converted into 3-HP by malonate reductase and 3-HP dehydrogenase, and glycerol may be converted into 3-HP by glycerol dehydratase and aldehyde dehydrogenase, a method for converting glucose or glycerol into 3-HP using modified E. coli has been well known. A dehydration reaction of glycerol, which is a reaction accompanied with radicals similarly to the transamination reaction, essentially requires coenzyme B12 for performing the reaction in the presence of oxygen.
In view of industrial fermentation, since it is difficult to use a coenzyme such as coenzyme B12 as a material of a culture medium due to its cost, and microorganisms such as yeast may not biosynthesize or absorb the corresponding material in cells, the β-alanine pathway or glycerol pathway is not suitable as the metabolic pathway for producing 3-HP using yeast. Recently, research modifying the key enzymes for overcoming this problem has been reported. [U.S. Pat. No. 7,655,451 B2]
Malonyl-CoA is synthesized from acetyl-CoA in the cytosol, and can thereby be reduced to 3-HP. In the case of bacteria such as E. coli, acetyl-CoA is formed from pyruvate in the cytosol, and can thereby be used as a substrate of the TCA cycle or other metabolic reaction. However, as described above, in yeast having independent sub-cellular compartments, generally, acetyl-CoA is synthesized in the mitochondria and is used as a substrate of the TCA cycle, and acetyl-CoA in the cytosol is produced via acetate producing reaction, which is a side-reaction of an ethanol production reaction, or a citric acid circulation reaction. All of the reactions of producing acetyl-CoA from acetate or citric acid are reactions consuming ATP, and since yeast further consumes energy in order to obtain acetyl-CoA in cytosol as compared to bacteria, yeast may be disadvantageous in view of energetics.
In yeast, environments such as the reduction state of the cytosol, folding after protein synthesis, codon usage, and the like, are different from those in bacteria, such that at the time of expressing an exogenous enzyme derived from bacteria, an activity thereof may not be exhibited or the activity may be significantly decreased. In addition, since the activity may be significantly changed by the presence or absence of oxygen or other metal ions, even in the case of exogenous enzymes having the same functions, expression results thereof in yeast may be different according to the origins of the enzymes. Actually, in the case of xylose isomerase (XI), which is an important enzyme of xylose metabolism, at the time of expressing XI derived from bacteria in yeast, mostly, an activity thereof was significantly low, but it was shown that yeast was successfully modified so as to perform xylose metabolism at a relatively high activity by introducing XI derived from anaerobic fungus. Thus, in the case of introducing a metabolic pathway derived from bacteria or Archaea such as the malonyl-CoA pathway in yeast, a gene having a high activity should be secured through genes performing the same functions with various origins.
There are various papers and patents associated with a method of genetically modifying Saccharomyces cerevisiae among various yeast strains to produce 3-HP, but in the case of Saccharomyces cerevisiae, since 3-HP is produced by the [pyruvic acid→acetaldehyde→acetic acid→acetyl-CoA→malonyl-CoA→malonate semialdehyde→3-HP] pathway, there are problems that this metabolic pathway to produce 3-HP is complicated, and productivity of 3-HP is relatively low (US 2010/0248233A1; Y. Chen et al., Metabolic Engineering, 22:104-109, 2014).
Accordingly, as a result of an effort to solve the above-mentioned problems, in order to overcome the disadvantage of yeast where ATP is consumed in a process of obtaining acetyl-CoA in cytosol, the present inventors conceived a shorter metabolic pathway of [Pyruvate→Acetaldehyde→Acetyl-CoA→Malonyl-CoA→Malonate semialdehyde→3-HP], which is directly converting acetaldehyde into acetyl-CoA without passing through an acetate intermediate, and confirmed that in the case of using a recombinant yeast comprising this pathway, unlike the case of using E. coli, not only the use of pH adjusting materials is decreased, and thereby production of salts is decreased, but also 3-HP may be produced from biomass at a high concentration and a high yield even at a low pH, thereby completing the present invention.